Restoring expression of tumour suppressor PTEN by engineered circular RNA‐enhanced Osimertinib sensitivity in non‐small cell lung cancer

This study provides a new strategy to construct circular RNA (circRNA) in vitro named NeoAna, with splicing sites concealed in CVB3 _ IRES. Re-storing phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression by engineered circRNA enhances sensitivity to Osimertinib in non-small lung cancer (NSCLC). Previous Anabaena permuted intron-exon system could

The well-known tumour suppressor, PTEN is a negative regulator of epidermal growth factor receptor (EGFR) signalling pathway, 4 and PTEN protein expression is often lost in lung cancer. 5Thus, restoring PTEN expression might reverse EGFR-TKI resistance. 6We synthesised PTEN protein template with NeoAna system (Figure S3B-E) and long-lasting PTEN protein expression was observed.Osimertinib-resistant cells were established in HCC827 and PC9 cells, since they harbour EGFR exon 19 deletion (Figure S4A-E).
The cEGFP_NeoAna was encapsulated by lipid nanoparticles (LNP) to form the stable complex and observed under electron microscope (Figure S6A).The average diameter of LNP_ cEGFP_NeoAna was measured to be 90.6 nm.The LNP encapsulation rates of cEGFP_NeoAna and cPTEN_NeoAna are 91.8% and 90.6%, respectively.Then, we transfected LNP_cEGFP_NeoAna into A549 and H1299 cells, and the green fluorescence was observed (Figure S6C,D).The xenograft mice models were built with PC9OR cells and cPTEN_NeoAna was administered through intratumour injection (Figure 2G).Mice in the cPTEN_NeoAna group had lowest tumour volume, Ki-67 expression and p-AKT expression (Figure 2H-K).Besides, we found both LNP and cPTEN_NeoAna are non-toxic (Figures 2L and S4E).Thus, these data demonstrated that cPTEN_NeoAna can effectively increase sensitivity to Osimertinib of NSCLC cells in vitro and in vivo.
We synthesised m1ψ-PTEN, cPTEN_Ana and cPTEN_NeoAna (Figure S2G) and treated cells with these RNAs in combination with Osimertinib.Then, CCK-8 and colony formation assays showed that cPTEN_NeoAna was more effective than cPTEN_Ana and m1ψ-PTEN (Figure 3A-D), although with no significant statistical difference.The apoptosis rate was highest in cPTEN_NeoAna group and increased along with Osimertinib concentration (Figure 3E,F).Taken together, these results illustrated that cells with cPTEN_NeoAna plus Osimertinib had lowest proliferation ability and highest apoptosis rates compared with other groups.Then, mouse model bearing xenograft tumour was established to assess the therapeutic efficacy of m1ψ-PTEN, cPTEN_Ana and cPTEN_NeoAna in vivo (Figure 3G).We observed that the tumour volume of cPTEN_NeoAna is smallest among four groups, which was in line with in vitro experiments (Figure 3H-J).The results of immunohistochemistry illustrated that PTEN was successfully restored in the three groups (Figure 3K).The expression of p-AKT and Ki-67 was lowest in cPTEN_NeoAna.We found that LNP and engineered RNA are non-toxic (Figure S6G).
Compared with PC9 and HCC827 cells, PTEN protein was slightly decreased in PC9OR and HCC827OR cells.When PTEN protein expression was restored, p-AKT and Kirsten rats arcomaviral oncogene homolog (KRAS) were significantly down-regulated (Figure 4A).Simultaneously, the expression of p-AKT but not KRAS decreased in an Osimertinib concentration-dependent manner.Then, we  S2 and S3; Figures 4C,D and S7).Herein, we found that there were 64 common genes between the up-regulated differentially expression genes (DEGs) of PC9OR versus PC9 and the down-regulated DEGs of PC9OR_ cPTEN_NeoAna versus PC9OR (Figure 4E).Glutathione peroxidase 2 (GPX2), transient receptor potential cation channel subfamily V member 4 (TRPV4) and aldo-keto reductase family 1 member C2 (AKR1C2) drew our attention.The RT-qPCR results showed that only the expression of AKR1C2 was in line with RNA-seq (Figure 4F,G).Compared with treatment-naïve cells, AKR1C2 protein was increased in PC9OR and HCC827OR cells and subsequently decreased when transfected with cPTEN_NeoAna (Figure 4H,I).Coimmunoprecipitation assays revealed that AKR1C2 could be immunoprecipitated by PTEN protein and vice versa (Figure 4J).Previous study reported that AKR1C2 could promote drug resistance of cancer cells by eliminating reactive oxygen species (ROS). 7Accordingly, we found that ROS level increased significantly after cPTEN_NeoAna treatment in PC9OR and HCC827OR cells but not Osimertinib treatment (Figures 4K,L and S8).
Besides, our strategy also successfully restored PTEN expression in colon cancer cells, DLD1 and DLD1 PTEN −/− (Figure S9A).CCK-8 and colony formation assays showed that the proliferative ability of DLD1 PTEN −/− cells was compromised after restoring PTEN expression with cPTEN_NeoAna (Figure S9B,C).
Overall, our NeoAna circRNA platform could synthesise circRNA without scar sequences in the final products.Restoring PTEN expression with NeoAna could be a promising strategy to overcome Osimertinib resistance in NSCLC.Therefore, NeoAna circRNA platform may be an alternative choice for mRNA and can be widely used in RNA-based therapeutics.

A U T H O R C O N T R I B U T I O N S
Mantang Qiu contributed to the study conception and design.Material preparation, data collection and analysis were performed by Haoran Li, Zheng Liu, Shaoyi Chen, Jingsheng Cai and Peiyu Wang.Haoran Li and Mantang Qiu wrote and revised the manuscript.Mantang Qiu and Kezhong Chen provided funding for this manuscript.All authors read and approved the final manuscript.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
M.Q., H.L., Z.L., and J.C. have applied for the patent (202310162756.5)related to the NeoAna system.All other authors declared no competing interests.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request.

E T H I C S S TAT E M E N T
This study was approved by the Ethics Committee of Peking University People's Hospital (2022PHE073).Studies on animals were conducted in accordance with relevant guidelines and regulations and were approved by the Animal Research Ethics Committee of Peking University People's Hospital.All methods were carried out in accordance with relevant guidelines and regulations.

F I G U R E 1
The design of group I permuted intron-exon splicing systems of NeoAna.(A) The schematic graph of Ana (Anabaena).(B) The schematic graph of NeoAna.(C) The results of agarose gel electrophoresis showed cEGFP_NeoAna.(D) The PCR amplification products of splicing sites.(E) The arrow directly showed the splicing site.(F) Western blots showed the expression of green fluorescent protein (GFP).(G) Western blots showed the expression of GFP in each peak of high-performance liquid chromatography (HPLC).(H) cEGFP_NeoAna expression in H1299 and 293 cells.(I) The schematic graph of HPLC of cEGFP_NeoAna.(J) The results of agarose gel electrophoresis showed the HPLC of cEGFP_NeoAna.(K) GFP of each peak of HPLC in H1299 and 293 cells.(L) The makers expression of innate immunity (**p < .01).

F
I G U R E 2 cPTEN_NeoAna can elevate non-small cell lung cancer cells to Osimertinib sensitivity.CCK-8 results showed cells with cPTEN_NeoAna is more sensitive to Osimertinib in PC9 Osimertinib-resistance (PC9OR) (A) and HCC827 Osimertinib-resistance (HCC827OR) cells (B).Colony formation assays showed that cells with cPTEN_NeoAna is more sensitive to Osimertinib in PC9OR (C) and HCC827OR (D).The apoptosis rate is increasing by the transfection of cPTEN_NeoAna in PC9OR (E) and HCC827OR (F).(G) The schematic graph of animal experiments.(H) The growth curve of tumour volume in different four groups.(I and J) Lipid nanoparticle (LNP) + cPTEN_NeoAna had smallest tumour volumes.(K) The representative photos of hematoxylin-eosin (HE), phosphatase and tensin homologue deleted on chromosome 10 (PTEN), Protein Kinase B (AKT), p-AKT and Ki-67.(L) The representative photographs of main organ.performed RNA-seq to reveal the potential mechanisms of PTEN enhancing Osimertinib sensitivity (Tables

F I G U R E 3
The efficacy of cPTEN_NeoAna to elevate non-small cell lung cancer (NSCLC) to Osimertinib sensitivity is slightly superior to cPTEN_Ana and m1ψ-PTEN.CCK-8 results showed the proliferative ability of cells of cPTEN_NeoAna is slightly inferior to those of cPTEN_Ana and m1ψ-PTEN in HCC827 Osimertinib-resistance (HCC827OR) (A) and PC9 Osimertinib-resistance (PC9OR) cells (B).Colony formation assays (C and D) also confirmed that finding.The apoptosis rate is highest in cells of cPTEN_NeoAna compared with those of cPTEN_Ana and m1ψ-PTEN in HCC827OR (E) and PC9OR (F).(G) The schematic graph of animal experiments.(H and J) cPTEN_NeoAna had smallest tumour volumes.(I) The growth curve of tumour volume in different four groups.(K) The representative photos of HE, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), AKT, p-AKT and Ki-67.
This work was supported by the National Key R&D Program of China (2023YFF0723500), the National Natural Science Foundation of China (92059203 and 82173386), the Beijing Nova Program (20230484314), the Research Unit of Intelligence Diagnosis and Treatment in Early Nonsmall Cell Lung Cancer (Chinese Academy of Medical Sciences, 2021RU002), the Peking University People's Hospital Research and Development Founds (RZ2022-04 and and the Peking University Medicine Sailing Program for Young Scholars' Scientific & Technological Innovation (BMU2023YFJHMX010 and BMU2023YFJHM X012).